Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
  • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters

Definitions

• the present invention relates to an oxidoreductase, to a fragment and an isolated DNA sequence of said oxidoreductase, to a fusion protein based on said oxidoreductase or said fragment and to a method for enantioselectively obtaining S-2-hydroxy acid esters.
• 2-Hydroxy acids and esters thereof are important chiral basic synthetic building blocks from which a number of compounds can be derived while preserving the chirality at the C2 atom, for example epoxides, alkyl esters, hydrazinyl esters, alpha-N-alkoxyamino esters or alpha-amino esters.
• the enzyme-catalyzed methods currently comprise three different methods.
• One route is the oxynitrilase-catalyzed synthesis of chiral cyanohydrins and subsequent hydrolysis thereof which is frequently also enzyme-catalyzed (Biotransformations in Organic Chemistry, A Textbook. 4th edition, Springer (2000), K. Faber; Cyanohydrin formation).
• This method has the disadvantage of using the toxic HCN.
• Another method is the resolution of 2-hydroxy acid esters with the aid of lipases, for example from Pseudomonas fluorescens (J. Org. Chem. 55, 812-815 (1991), Kinetic Resolution of 2-substituted Esters catalysed by a Lipase Ex. Pseudomonas fluoreszens, Kalaritis, P. et al.).
• the disadvantage of this method is the theoretical yield of only 50%.
• Another method is the synthesis of chiral 2-hydroxy acids and esters thereof by reducing prochiral 2-oxo acids or esters thereof. Transformation with whole yeast cells or with cells of Proteus vulgaris or Proteus mirabilis or methods using isolated enzymes is known. In the case of transformations with whole yeast cells, the reducing enzyme action on 2-oxo acids was attributed to the enzymes lactate dehydrogenase or malate dehydrogenase (Ramesh N. Patel Stereoselective Biocatalyse, NY (2000), 14.
• methods which comprise regenerating the coenzyme NAD with formate dehydrogenase, for example from Candida boidinii or else in recombinant form from Pseudomonas fluoreszens (Biotechnology, Biotransformations I (Rehm and Reed) 9. Alcohol Dehydrogenases-Characteristics, Design of Reaction Conditions J. Peters, WILEY-VCH-Verlag, (1998)).
• the method of enzymatically preparing R-2-hydroxy-4-phenylbutyric acid with the aid of D-lactate dehydrogenase from Staphylococcus epidermidis is mentioned here by way of example (Industrial Biotransformations, Liese, K. Seelbach, C.
• a disadvantage of coenzyme regeneration using formate dehydrogenase is the low specific activity of said formate dehydrogenase (4 to 10 U/mg) and the high costs of preparing the enzyme. From an economic viewpoint it is therefore necessary to use the enzyme several times, resulting in a comparatively substantially more complex and thus more expensive process control.
• this object is achieved by an oxidoreductase reducing 2-oxo acid esters to the corresponding S-2-hydroxy acid esters in the presence of NADPH and water.
• the invention relates, inter alia, to oxidoreductases which may be obtained from Lactobacillus (L.) reuteri, L. kefiri, L. kandleri, L. parabuchneri, L. cellobiosus or L. fermentum , for example.
• the invention further relates to the Lactobacillus reuteri oxidoreductase which has the DNA sequence according to SEQ ID NO: 19 and the amino acid sequence according to SEQ ID NO: 18 as described in the attached sequence listing.
• the invention further relates to an oxidoreductase wherein more than 70% of the amino acids therein are identical to the amino acid sequence SEQ ID NO: 18 and which has a specific activity of more than 1 mmol per mg, based on the conversion of ethyl 2-oxo-4-phenylbutyrate to ethyl S-2-hydroxy-4-phenylbutyrate.
• the specific activity of the oxidoreductase according to SEQ ID NO: 18 or of its derivatives or analogs is measured using the assay system described in example 2.
• the invention further relates to an oxidoreductase which has from 1 to 50 amino acids more or from 1 to 50 amino acids fewer than the oxidoreductase having the amino acid sequence SEQ ID NO: 18 and a specific activity of more than 1 ⁇ mol per mg, based on the conversion of ethyl 2-oxo-4-phenylbutyrate to ethyl S-2-hydroxy-4-phenylbutyrate.
• Preference is given to oxidoreductases which have from 1 to 25 amino acids, in particular from 2 to 20 amino acids, preferably from 3 to 10 amino acids, more or fewer than occur in the amino acid sequence of SEQ ID NO: 18.
• the invention further relates to an oxidoreductase which has the amino acid sequence of SEQ ID NO: 18 and has been modified once, twice, three, four or five times by a water-soluble polymer and has a specific activity of more than 1 mmol per mg, based on the conversion of ethyl 2-oxo-4-phenylbutyrate to ethyl S-2-hydroxy-4-phenylbutyrate.
• a water-soluble polymer is polyethylene glycol.
• Polyethylene glycol is preferably bound to the N-terminal end of the oxidoreductase according to SEQ ID NO: 18.
• the oxidoreductase according to SEQ ID NO: 18 may also be bound to a solid such as polyethylene, polystyrene, polysaccharide, cellulose or cellulose derivative.
• the invention further relates to an oxidoreductase fragment which represents a fragment of the amino acid sequence SEQ ID NO: 18, having from 5 to 30 amino acids.
• a fragment of SEQ ID NO: 18 which has an amino acid chain of from 6 to 25 amino acids, in particular from 8 to 20 amino acids, preferably from 10 to 18 amino acids, in particular the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2. Fragments of this kind may be used, for example, for finding the inventive oxidoreductase from L. reuteri or from any other microorganisms.
• the invention further relates to a fusion protein which represents the oxidoreductase having the amino acid sequence SEQ ID NO: 18 or a fragment thereof having from 5 to 30 amino acids and said oxidoreductase or said fragment thereof being linked at the N terminus or carboxy terminus via a peptide bond to another polypeptide.
• Fusion proteins can be removed relatively easily from other proteins, for example, or are expressed in relatively large quantities in the cells.
• the invention further relates to an antibody which binds specifically to the oxidoreductase according to SEQ ID NO: 18 or to a fragment thereof according to SEQ ID NO: 1 or SEQ ID NO: 2.
• These antibodies are prepared according to known methods by immunizing suitable mammals such as horse, mouse, rat or pig and subsequently obtaining said antibodies.
• the antibodies may be monoclonal or polyclonal.
• the invention also relates to an isolated nucleic acid sequence which codes for the oxidoreductases according to SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 2.
• the invention further relates to an isolated deoxyribonucleic acid sequence (DNA sequence) of the oxidoreductase catalyzing the reduction of 2-oxo acid esters to corresponding S-2-hydroxy acid esters in the presence of NADPH and water, wherein said DNA sequence is selected from the group consisting of
• Hybridization is described, for example, by Sambrook and Russel in Molecular Cloning a laboratory Manual , volume 1, chapter 1, protocol 30-32.
• the invention further relates to an isolated DNA sequence wherein more than 70% of the nucleic acid bases are identical to the DNA sequence according to SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 19 or to the complementary strands thereof and which encodes a protein having a specific activity of more than 1 ⁇ mol per mg, based on the conversion of ethyl 2-oxo-4-phenylbutyrate to ethyl S-2-hydroxy-4-phenylbutyrate.
• DNA sequences wherein from 80% to 99.5%, in particular from 90% to 99.5%, preferably from 99% to 99.5%, of the nucleic acid bases are identical to the DNA sequence according to SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 19.
• the invention further relates to an isolated DNA sequence having from 10 to 50 nucleic acid bases and a sequence corresponding to part of a DNA sequence according to SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 19 or to the complementary strand thereof.
• Preference is given to a nucleic acid sequence having from 15 to 45 nucleic acid bases, in particular from 20 to 40 bases, particularly preferably from 30 to 40 nucleic acid bases.
• the nucleic acid sequences mentioned are suitable as molecular samples or as primers for the polymerase chain reaction (PCR).
• the invention further relates to a cloning vector comprising one or more of the nucleic acid or DNA sequences mentioned above.
• the invention further relates to an expression vector which is present in a bacterial, yeast, insect, plant or mammalian cell and which comprises one or more of the nucleic acid or DNA sequences mentioned above and which is linked in a suitable manner to an expression control sequence.
• the invention further relates to a host cell which is a bacterial, yeast, insect, plant or mammalian cell and which has been transformed or transfected with any of the abovementioned expression vectors.
• the homologies of the abovementioned DNA sequences or amino acid sequence are calculated by adding up the number of amino acids or nucleic acid bases identical to partial sequences of the respective proteins or DNA sequences, dividing this by the total number of amino acids or nucleic acid bases and multiplying by one hundred.
• cloning vectors examples include ppCR-Script, pCMV-Script, pBluescript (Stratagene), pDrive cloning Vector (Quiagen), pS Blue, pET Blue, pET LIC vectors (Novagen) and TA-PCR cloning vectors (Invitrogen).
• Suitable expression vectors are pKK223-3, pTrc99a, pUC, pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRMl/pRM9, pTrxFus, pAS1, pGEx, PMAL, pTrx).
• trp-lac (tac) promoter examples include trp-lac (tac) promoter, trp-lac (trc) promoter, lac promoter, T7 promoter, XpL promoter.
• the Lactobacillus reuteri oxidoreductase is a homodimer having a molecular weight of from 30 to 35 kDa, as determined in an SDS gel, and a molecular weight of from 60 to 65 kDa, as determined by gel permeation chromatography.
• the optimal temperature is in the range from 55° C. to 60° C. and its optimal pH is from 6.5 to 7.0.
• Lactobacillus reuteri oxidoreductase has good temperature and pH stabilities and is stable for at least 5 hours within a pH range from 4.5 to 8.5 and a temperature range from 15° C. to 50° C. and furthermore exhibits high stability in organic solvents.
• the enzyme can be isolated in particular from microorganisms of the genus Lactobacillus and detected in a spectrophotometric assay via the decrease in NADPH at 340 nm in the presence of an appropriate substrate, for example ethyl 2-oxo-4-phenylbutyric acid or ethyl 2-oxovaleric acid.
• an appropriate substrate for example ethyl 2-oxo-4-phenylbutyric acid or ethyl 2-oxovaleric acid.
• the Lactobacillus reuteri oxidoreductase of the invention was cloned and overexpressed in Escherichia (E.) coli , with activities of from 10 000 U/g to 30 000 U/g of E. coli wet weight.
• the enzyme is thus inexpensive and available in large quantities. No related sequences were found in databases, and only a distant relationship to enzymes of the group of hydroxyacyl-CoA dehydrogenases might be suspected.
• the invention also relates to a method for obtaining Lactobacillus reuteri oxidoreductase.
• the DNA coding for Lactobacillus reuteri oxidoreductase is expressed in a suitable prokaryotic or eukaryotic microorganism.
• a suitable prokaryotic or eukaryotic microorganism Preference is given to Lactobacillus reuteri oxidoreductase being transformed into and expressed in an Escherichia coli strain, in particular Escherichia coli BL21star (DE3) cells (Invitrogen, cat. No. C6010-03, derived from E. coli BL21, with a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter, without ompT and Lon protease, B121 star has mutation in RNaseE (rnel31).
• Lactobacillus reuteri oxidoreductase can be obtained, for example, by culturing the recombinant Escherichia coli cells, inducing expression of said oxidoreductase and subsequently, after approximately 10 to 18 hours (h), disrupting said cells by ultrasound treatment or by wet grinding with glass beads in a ball mill (Retsch, 10 min, 24 Hz).
• the cell extract obtained may either be used directly or be purified further.
• the cell extract is centrifuged, for example, and the supernatant obtained is subjected to hydrophobic interaction chromatography, for example hydrophobic interaction chromatography on Butyl Sepharose Fast Flow (Pharmacia) and subsequent gel permeation (Superdex 200 HR, Pharmacia).
• hydrophobic interaction chromatography for example hydrophobic interaction chromatography on Butyl Sepharose Fast Flow (Pharmacia) and subsequent gel permeation (Superdex 200 HR, Pharmacia).
• the invention further relates to a method for enantioselectively obtaining S-2-hydroxy acid esters, which comprises reducing 2-oxo acid esters in the presence of oxidoreductase, NADPH and water to the corresponding S-2-hydroxy acid ester and isolating the S-2-hydroxy acid ester produced.
• the method of the invention has a long useful life, an enantiomeric purity of more than 94% of the chiral S-2-hydroxy acid esters prepared and a high yield based on the amount used of the 2-oxo acid ester.
• NADPH means reduced nicotinamide adenine dinucleotide phosphate.
• NADP means nicotinamide adenine dinucleotide phosphate.
• 2-oxo acid esters means, for example, compounds of the formula I R2—C(O)—C(O)—O—R1 (I).
• R1 is
• S-2-hydroxy acid esters means compounds of the formula II R2—C(OH)—C(O)—O—R1 (II) where the —OH group is in S configuration with respect to the carbon atom to which it is bound and R1 and R2 are as defined in formula I.
• aryl means aromatic carbon radicals having from 6 to 14 ring carbons.
• —(C 6 -C 14 )-aryl radicals are phenyl, naphthyl, for example 1-naphthyl, 2-naphthyl, biphenylyl, for example 2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl or fluorenyl.
• Preferred aryl radicals are biphenylyl radicals, naphthyl radicals and in particular phenyl radicals.
• halogen means an element of the series fluorine, chlorine, bromine and iodine.
• —(C 1 -C 20 )-alkyl means a hydrocarbon radical whose carbon chain is straight-chain or branched and comprises from 1 to 20 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonenyl or decanyl.
• —(C 3 -C 7 )-cycloalkyl means cyclic hydrocarbon radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
• —(C 5 -C 14 )-heterocycle is a monocyclic or bicyclic 5-membered to 14-membered heterocyclic ring which is partially or completely saturated. Examples of heteroatoms are N, O and S.
• Examples of the terms —(C 5 -C 14 )-heterocycle are radicals deriving from pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, 1,2,3,5-oxathiadiazole-2-oxides, triazolones, oxadiazolones, isoxazolones, oxadiazolidindiones, triazoles, substituted by F, —CN, —CF3 or —C(O)—O—(C 1 -C 4 )-alkyl, 3-hydroxypyrro-2,4-diones, 5-oxo-1
• Examples of preferred compounds of the formula I are ethyl 2-oxovalerate, ethyl 2-oxo-4-phenylbutyrate, ethyl pyruvate, ethyl phenylglyoxylate, ethyl 2-oxo-3-phenylpropionic acid, ethyl 8-chloro-6-oxooctanoate, ethyl 2-oxobutyrate, ethyl 2-oxohexanoate, methyl phenylglyoxylate, methyl 2-oxovalerate, methylpyruvate, methyl 2-oxo-4-phenylbutyrate, methyl 2-oxo-3-phenylpropionic acid, methyl 8-chloro-6-oxooctanoate, methyl 2-oxobutyrate and methyl 2-oxohexanoate.
• S-2-hydroxy acid esters are, for example, ethyl S-2-hydroxyvalerate, ethyl S-2-hydroxy-4-phenylbutyrate, ethyl L-lactate or ethyl S-mandelate.
• Suitable oxidoreductases are derived from Lactobacillus reuteri , for example. It is possible to use in the method of the invention either a completely or partially purified oxidoreductase or an oxidoreductase contained in cells.
• the cells used in this connection may be in native, permeabilized or lysed form. Preference is given to using the cloned oxidoreductase according to SEQ ID NO: 18.
• the volume activity of the oxidoreductase used is from 250 units/ml (U/ml) to 20 000 U/ml, preferably approximately 4 000 U/ml. From 5 000 to 250 000 U, preferably approximately 10 000 U to 50 000 U, of oxidoreductase per kg of compound of the formula I to be converted are used.
• the enzyme unit 1 U in this connection corresponds to the amount of enzyme required in order to convert 1 mmol of ethyl 2-oxo-phenylbutyrate to ethyl S-2-hydroxy-4-phenylbutyrate per minute (min).
• the invention further relates to a method for enantioselectively obtaining S-2-hydroxy acid ester, which comprises
• Suitable dehydrogenases are alcohol dehydrogenases from Thermoanaerobium brockii, Lactobacillus kefir or Lactobacillus brevis , said enzymes requiring the coenzyme NADPH (DE 19 610 984, EP 0 456 107, WO 97/32012).
• Suitable cosubstrates of the alcohol dehydrogenase used are alcohols such as ethanol, 2-propanol (isopropanol), 2-butanol, 2-pentanol or 2-octanol.
• NADP may also be carried out using the known enzymes used for regenerating NADPH, for example glucose dehydrogenase or NADPH-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol. Bioeng. [1999] 64, 187-193, Pilot-scale production and isolation of recombinant NAD and NADP specific formate dehydrogenase).
• enzymes used for regenerating NADPH for example glucose dehydrogenase or NADPH-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol. Bioeng. [1999] 64, 187-193, Pilot-scale production and isolation of recombinant NAD and NADP specific formate dehydrogenase).
• the cosubstrate suitable for the method of the invention when using glucose dehydrogenase is glucose.
• suitable cosubstrates of formate dehydrogenase are salts of formic acid such as ammonium formate, sodium formate or calcium formate.
• Lactobacillus minor alcohol dehydrogenase (DE 101 19274). It is possible to use in the method of the invention either completely or partially purified alcohol dehydrogenase or whole cells containing said alcohol dehydrogenase.
• the cells used in this connection may be in native, permeabilized or lysed form. From 10 000 U to 200 000 U, preferably approximately 25 000 U to 100 000 U, of alcohol dehydrogenase are used per kg of compound of the formula I to be converted.
• the enzyme unit 1 U in this connection corresponds to the amount of enzyme required in order to convert 1 ⁇ mol of the cosubstrate (e.g. 2-propanol) per minute (min).
• a buffer for example potassium phosphate buffer, Tris/HCl buffer or triethanolamine buffer, having a pH of from 5 to 10, preferably from 6 to 9.
• the buffer concentration is from 10 mM to 150 mM, preferably from 90 mM to 110 mM, in particular 100 mM.
• the buffer may also contain ions for stabilizing or activating both enzymes, for example magnesium ions for stabilizing Lactobacillus minor alcohol dehydrogenase.
• the temperature in the methods of the invention is, for example, from approximately 10° C. to 60° C., preferably from 30° C. to 55° C.
• the invention further relates to a method for enantioselectively obtaining S-2-hydroxy acid ester, which comprises
• Examples of preferred organic solvents are diethyl ether, tert-butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane and cyclohexane.
• the reaction mixture comprises an aqueous phase and an organic phase when additional solvents are used.
• the organic phase is formed by a suitable solvent in which the substrate has been dissolved or by the water-insoluble substrate itself.
• the organic phase is from about 5% to 80%, preferably from 10% to 40%, of the total reaction volume.
• water forms the second liquid phase.
• a solid or another liquid phase which is produced, for example, by incompletely dissolved oxidoreductase and/or alcohol dehydrogenase or by the compound of the formula I may additionally also be present.
• preference is given to two liquid phases without solid phase.
• Preference is given to mixing said two liquid phases mechanically, so as to generate large surface areas between the two liquid phases.
• the concentration of the cofactor NADPH is from 0.001 mM to 0.1 mM, in particular from 0.005 mM to 0.02 mM, based on the aqueous phase.
• suitable stabilizers are glycerol, sorbitol or dimethyl sulfoxide (DMSO).
• the compounds of the formula I are used in the method of the invention in an amount of from 10% to 60%, preferably from 15% to 50%, in particular from 20% to 40%, based on the total volume.
• the amount of cosubstrate for regenerating NADP to NADPH, such as isopropanol, is from about 5% to 50%, preferably from 10% to 30%, in particular from 15% to 25%, based on the total volume.
• the method of the invention is carried out, for example, in a closed reaction vessel made of glass or metal.
• the components are individually transferred to said reaction vessel and stirred under an atmosphere of nitrogen or air, for example.
• the reaction time is from 1 hour to 48 hours, in particular from 2 hours to 24 hours, depending on the substrate and the compound of the formula I used.
• the reaction mixture is worked up.
• the aqueous phase is removed and the organic phase is filtered.
• the aqueous phase may be extracted once more and, like the organic phase, worked up further.
• This is followed by evaporating the solvent from the clear organic phase, where appropriate.
• This results, for example, in the product ethyl S-2-hydroxy-4-phenylbutyrate which is more than 94% enantiomerically pure and essentially free of the reactant ethyl 2-oxo-4-phenylbutyrate.
• the total yield of the process is from 50% to 95%, based on the amount of reactant used.
• 125 mg of cells were then resuspended with 800 ⁇ l of disruption buffer (100 mM triethanolamine (TEA), pH 7.0), mixed with 1 g of glass beads and disrupted in a ball mill (Retsch) at 4° C. for 10 min. The supernatant obtained after 2 minutes (min) of centrifugation at 12 000 revolutions per minute (rpm) was used in activity screening and for determining the enantiomeric excess.
• the substrates used were ethyl 2-oxopentanoate and ethyl 2-oxo-4-phenylbutyrate.
• reaction mixes for ee determination were extracted with chloroform after 24 hours (h) and the enantiomeric excess was analyzed by means of GC.
• DSMZ stands for Deutsche Sammlung für Mikroorganismen und Zellkulturen, Mascheroder Weg lb, 38124 Braunschweig, Germany.
• Table 1 reveals that a plurality of species of the genus Lactobacillus have an NADPH-dependent oxidoreductase with ethyl 2-oxo-4-phenylbutyrate or ethyl 2-oxopentanoate as substrate.
• the crude extract obtained after centrifugation was then adjusted to a final concentration of 50% ammonium sulfate by adding 242 mg of (NH 4 ) 2 SO 4 and stirred at 4° C. for 1 h.
• the pellet was then removed by centrifugation at 12 000 rpm for 10 min and the supernatant obtained was further purified by means of FPLC.
• the enzyme was purified using hydrophobic interaction chromatography on Butyl Sepharose Fast Flow (Pharmacia) and subsequent gel permeation (Superdex 200 HR, Pharmacia).
• the enzyme was eluted at 0 M (NH 4 ) 2 SO 4 .
• the active fractions were combined and reduced to a suitable volume by means of ultrafiltration (cut-off 10 kDa).
• the enzymic activity of oxidoreductase is determined in the assay system according to example 1 (activity screening mix) and the amount of protein was determined according to Lowry et al.
• the specific activity is the enzyme activity divided by the amount of protein, with 1 unit (U) corresponding to the conversion of 1 mmol per min.
• the molecular weight standards used were catalase (232 kDa), aldolase (158 kDa), albumin (69.8 kDa) and ovalbumin (49.4 kDa).
• the enzyme preparation was fractionated in a 10% strength sodium dodecyl sulfate (SDS) gel and transferred to a polyvinylidene difluoride membrane (PVDF membrane).
• SDS sodium dodecyl sulfate
• PVDF membrane polyvinylidene difluoride membrane
• SDS-PAGE band of the same protein was reduced with dithiothreitol, carboxymethylated and digested with endoproteinase Lys-C.
• Chromosomal DNA was extracted from Lactobacillus reuteri cells according to the method described in “Molecular cloning” by Maniatis & Sambrook.
• the resulting genomic DNA served as template for the direct polymerase chain reaction (PCR) using degenerated primers.
• Said degenerated 5′ primers were derived from the N-terminal amino acid sequence (Seq. No: 1) and the 3′ primers were derived from the amino acid sequence of an internal peptide (Seq. No: 2), taking into account the universal gene code (Seq. No: 3, 4, 5 and 6).
• Primer constructs are listed below.
• the primers were prepared by known methods. 5′-Oligo 3: ATGAARAAYATYATGATYGCHGGCGC 5′-Oligo 4: ATGAARAAYATYATGATYGCHGGTGC 3′-Oligo 5: RTGHARATCMCGTTCRTAATC 3′-Oligo 6: RTGHARATCMCGTTCRTAGTC
• the amplification was carried out in PCR buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , 1 mM deoxynucleoside triphosphate mix (dNTP) mix, 30 pmol of each primer and 2.5 U of AmpliTaq Gold (Applied Biosystems)].
• PCR buffer 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , 1 mM deoxynucleoside triphosphate mix (dNTP) mix, 30 pmol of each primer and 2.5 U of AmpliTaq Gold (Applied Biosystems)].
• AmpliTaq Gold polymerase 10 min, 94° C.
• the subsequent 35 PCR cycles 94° C., 60 sec; 53° C., 45 sec; 72° C., 60 sec
• the reaction was cooled to 4° C. and the entire PCR mixture was applied to a 1%
• Sequence analysis of the 159 bp gene fragment revealed an open reading frame of 53 amino acids, which also included the sequence fragments of both the N terminus and the internal peptide.
• Chromosomal DNA from L. reuteri cells was digested with Eco RI endonuclease and used for religation with T4 ligase. The resulting circular, chromosomal DNA fragments served as template for the iPCR.
• the PCR signal was then amplified by a nesting PCR.
• the optimal MgCl 2 concentration was at 2 mM, with 2 ⁇ l of the first iPCR as template.
• a gradient PCR determined a temperature of 57° C. as being optimal for the primer pair 9 and 11.
• the following amplification cycles with AmpliTaq Gold DNA polymerase were required in order to be able to detect a specific PCR product of 1 200 bp in length: Cycle 1: 95° C., 10 min Cycle 2 ⁇ 40: 95° C., 45 s 57° C., 1 min 72° C., 90 s Cycle 3: 72° C., 7 min 4° C., ⁇
• the specific band of 1 200 bp in length was purified via a 1% agarose gel by means of the Qiaex gel extraction kit (Qiagen, Hilden, Germany) and used in a ligation reaction with the TA-PCR cloning vector pCR2.1 (Invitrogen).
• the sequence analysis of the 1 241 bp DNA fragment revealed an open reading frame of 148 amino acids in the 5′-terminal region.
• the sequence of the first 15 N-terminal amino acids corresponded to the C-terminal amino acid sequence Seq No: 7 of 4.2.
• the analysis of the C-terminal sequence of the 1 241 bp DNA fragment revealed enclosed regulatory DNA segments to the N-terminal end of the L. reuteri (S)-ADH gene.
• Chromosomal DNA from L. reuteri cells was digested with Af1 III endonuclease and used for religation with T4 ligase. The resulting circular chromosomal DNA fragments served as template for the iPCR.
• the PCR signal was then amplified by a nesting PCR.
• the optimal MgCl 2 concentration in this reaction was 2 mM, and 2 ⁇ l of the first iPCR were used as template for said nesting PCR.
• the following amplification cycles with AmpliTaq Gold DNA polymerase were required in order to be able to detect a specific PCR product of 950 bp in length: Cycle 1: 94° C., 10 min Cycle 2 ⁇ 40: 94° C., 45 s 56° C., 1 min 72° C., 1:45 min Cycle 3: 72° C., 7 min 4° C., ⁇
• a specific band of 950 bp in length was purified by means of the Qiaex gel extraction kit (Qiagen) via a 1% agarose gel and used in a ligation reaction with the TA-PCR cloning vector pCR2.1 (Invitrogen).
• the DNA fragment inserted into the pCR2.1 vector was 822 bp in length and had at the N terminus an open reading frame of 126 amino acids, which ended with a stop codon and a termination loop (Seq. No: 15).
• the 5′ peptide of 12 amino acids corresponded to the C-terminal end of sequence No: 12.
• the 822 bp DNA fragment generated by iPCR comprised the C-terminal end of the gene segment coding for an L. reuteri oxidoreductase.
• Genomic DNA of L. reuteri cells served as template for the polymerase chain reaction.
• the amplification was carried out in a PCR buffer [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO 4 ; 1 mM dNTP mix; 30 pmol of each primer and 2.5 U of Platinum Pfx DNA polymerase (Invitrogen)] with 300 ng of template and the following temperature cycles: Cycle 1: 94° C., 2 min Cycle 2 ⁇ 30: 94° C., 15 s 58° C., 30 s 68° C., 75 s Cycle 3: 68° C., 7 min 4° C., ⁇
• the resulting PCR product was digested with Nde I and Hind III and ligated into the pET21a vector (Novogene, Madison, USA) backbone treated with the same endonucleases. After transformation of 2 ⁇ l of the ligation mixture into E. coli Top 10 F′ cells, plasmid DNAs of ampicillin-resistant colonies were checked by means of restriction analysis with endonucleases Nde I and Hind III for correct ligation. The expression construct pET21-reut#10 was sequenced.
• the Lactobacillus reuteri oxidoreductase gene has an open reading frame of 882 bp in total (Seq. No: 19), corresponding to a protein of 294 amino acids (Seq. No: 18).
• Competent Escherichia coli StarBL21(De3) cells were transformed with the pET21-reut#10 expression construct containing the oxidoreductase gene.
• the strain was cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing ampicillin (50 ⁇ g/ml), until an optical density, measured at 500 nm, of 0.5 was reached.
• Oxidoreductase expression was induced by addition of isopropyl-thiogalactoside (IPTG) at a final concentration of 1 mM. After induction at 25° C. and 220 rpm for 8 hours, the cells were harvested and frozen at ⁇ 20° C.
• IPTG isopropyl-thiogalactoside
• the enzyme was expressed with activities of from 10 000 U/g to 30 000 U/g of wet weight E. coli .
• the enzyme is thus inexpensive and available in large quantities.
• the pH optimum was determined by determining the enzymic reaction in the particular buffer listed in table 3. A pH optimum of between 6.5 and 7 was determined for the enzyme of the invention. The enzyme has 80% of its maximum activity in the pH range from 4.5 to 8, with said activity then rapidly decreasing at pH values of below 4.0 and above 8.5.
• the recombinant L. reuteri oxidoreductase is stable in the pH range from 4.5 to 8.0 and may be incubated without loss of activity for at least 5 h. 50% and 40% remaining activity were found for pH 4.0 and 9.0, respectively, after 5 h. pH values above 9.5 lead to immediate inactivation of the enzyme.
• the optimal assay temperature was determined by measuring the enzyme activity in the temperature range from 15° C. to 70° C. in the standard measurement mixture. As table 5 indicates, the maximum activity of the enzyme is at 55° C., and rapidly decreases thereafter. TABLE 4 Temper- Activity Temper- Activity ature in U/ml of ature in U/ml of (° C.) undiluted enzyme (° C.) undiluted enzyme 15 385 45 2900 20 745 50 3200 25 1090 55 3800 30 1350 60 617 35 1860 65 180 40 2500 70 90 5.4 Temperature Stability
• the temperature stability for the range from 15° C. to 70° C. was determined in a manner similar to that described under 5.2. For this purpose, in each case a 1:200 dilution of the purified enzyme was incubated at the particular temperature for 60 min and 180 min and then measured at 30° C. using the above assay mixture.
• the enzyme is totally stable in a temperature range from 15° C. to 50° C. and exhibits no loss of activity whatsoever after 3 h of incubation. At 55° C., an enzyme activity is no longer detectable after only 30 min.
• the substrate spectrum of the oxidoreductase of the invention was determined by measuring the enzyme activity with a number of ketones, oxo acids and esters thereof. For this purpose, the standard measurement mixture (example 5.1) was used with different substrates. The activity with ethyl 2-oxo-4-phenylbutyrate was set to 100% and all other substrates were related thereto. The enzyme exhibited no NADP-dependent dehydrogenase activity for ethyl (R)- or (S)-2-hydroxy-4-phenylbutyrate, (R)- or (S)-4-chloro-3-hydroxybutyrate and (D)- or (L)-ethyl lactate.
• the ee value was determined by preparing the following reaction mixture for selected substrates. 100 ⁇ l NADPH (50 mM) 60 ⁇ l substrate (100 mM) and 1 to 2 units of oxidoreductase
• the recombinant Lactobacillus reuteri oxidoreductase reduces in particular 2-oxo acid esters stereoselectively to the corresponding 2-hydroxy acid esters.
• the corresponding 2-oxo acids were not accepted as substrates, methyl ketones were also hardly reduced at all, 3-oxo acid esters are partly reduced but mostly not stereoselectively.
• L. reuteri oxidoreductase is remarkably stable with respect to organic solvents. Furthermore, the enzyme is even stabilized in organic water-miscible and water-immiscible solvents, compared to incubation in pure buffer.
• the enzyme solution used was the 1:300 diluted lysate (600 ⁇ l) obtained from 0.1 g of recombinant E. coli cells.

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